Camostat

VeroE6 or HEK293T-hACE2 cells were seeded 4000 or 8000 cells/well of 96-well plate. After 24 h growth medium was replaced with increasing concentration of Imatinib (Novartis^®) and washed away after 8 h. Two days later cell viability was measured using the XTT cell proliferation kit (Biological Industries^®). Cells were treated with camostat (Tocris^®) and E64d (Caymanchem) in reported doses^{10 (link),15 ,63 (link),64 (link)}.

CMK furin inhibitor experiments: 293T cells were transfected with plasmids expressing Gag/pol, luciferase, and spike. Furin inhibitor CMK (Calbiochem) was added at either 5 M or 25 μM concentration three hours post transfection. The supernatants and cell lysates were collected after 48 hours for infectivity on target cells or for western blotting.
E64D and camostat experiments: ACE2 or ACE2 and TMPRSS2 transfected 293T cells were either E64D (Tocris) or camostat (Sigma-Aldrich) treated for 3 hours at each drug concentration before the addition of a comparable amount of input viruses pseudotyped with WT, H69/V70 deletion or VSV-G (approx. 1 million RLU). The cells were then left for 48 hours before addition of substrate for luciferase (Promega) and read on a Glomax plate reader (Promega). The RLU was normalized against the no-drug control which was set as 100%.

The reduction of luciferase gene expression was detected to evaluate the infection inhibition effect of hACE2 protein (expressed in HEK293 cells, Sino Biological), protease inhibitor Decanoyl-RVKR-CMK (Furin inhibitor, R&D Systems), protease inhibitor Camostat (TMPRSS2 inhibitor, Tocris Bioscience), protease inhibitor E64D (Cathepsin L inhibitor, APExBIO), monoclonal antibody, and sera. As described previously^{19 (link),20 (link)}, the tested samples were diluted (starting with 30 times dilution and three times gradient, a total of eight gradients), after which the virus solution was added. On each 96-well plate, eight virus control wells and eight cell control wells were arranged. In the virus control wells, no test sample but only virus solution was added; in the cell control wells, no virus solution but only a complete culture medium was added. The 96-well plates were incubated at 37 °C for 1 h, after which trypsinized Huh7 cells (2 × 10⁴/100 μL) were added to each well. After incubation for 24 h in an atmosphere comprising 5% CO₂ at 37 °C, luminescence was measured as described above. The EC₅₀ value of each sample was calculated using the Reed–Muench method.